Pichia pastoris secretes recombinant proteins less efficiently than Chinese hamster ovary cells but allows higher space-time yields for less complex proteins

Abstract

Chinese hamster ovary (CHO) cells are currently the workhorse of the biopharmaceutical industry. However, yeasts such as Pichia pastoris are about to enter this field. To compare their capability for recombinant protein secretion, P. pastoris strains and CHO cell lines producing human serum albumin (HSA) and the 3D6 single chain Fv-Fc anti-HIV-1 antibody (3D6scFv-Fc) were cultivated in comparable fed batch processes. In P. pastoris, the mean biomass-specific secretion rate (qp ) was 40-fold lower for 3D6scFv-Fc compared to HSA. On the contrary, qp was similar for both proteins in CHO cells. When comparing both organisms, the mean qp of the CHO cell lines was 1011-fold higher for 3D6scFv-Fc and 26-fold higher for HSA. Due to the low qp of the 3D6scFv-Fc producing strain, the space-time yield (STY) was 9.6-fold lower for P. pastoris. In contrast, the STY of the HSA producer was 9.2-fold higher compared to CHO cells because of the shorter process time and higher biomass density. The results indicate that the protein secretion machinery of P. pastoris is much less efficient and the secretion rate strongly depends on the complexity of the recombinant protein. However, process efficiency of the yeast system allows higher STYs for less complex proteins.

Keywords: CHO cells; Fed batch; Protein secretion; Recombinant protein production; Volumetric productivity.

Figure 1

Time courses of fed batch cultivations. Mean cell dry mass concentration, cell viability and product concentration of the (A) P. pastoris 3D6scFv-Fc, (B) P. pastoris HSA, (C) CHO 3D6scFv-Fc, and (D) CHO HSA cultivations. Arrows mark the feed start. Data represent mean values ± standard deviation of two independent cultivations (CHO HSA: data of one cultivation after 163 h). Curves for cell dry mass and product concentration were calculated using the smoothing spline algorithm of the Matlab Curve Fitting Toolbox. Biomass-specific secretion rates of (E) 3D6scFv-Fc producers and (F) HSA producers and space-time yields of (G) 3D6scFv-Fc producers and (H) HSA producers during the feed phase. Data were calculated using smoothed product concentrations of two independent cultivations. P. pastoris and CHO cells were cultivated in comparable DASGIP bioreactor systems. P. pastoris cultures were inoculated with an optical density (OD₆₀₀) of 3.0 and grown at 25°C, pH 5.85, and 20% dissolved oxygen. A constant feed rate of 2.38 g h^–1 fed batch medium (500 g L^–1 glucose) was applied for 113 h. The CHO cell concentrations at cultivation start were adjusted to 2.5 × 10⁵ cells mL^–1 and the cultures were grown at 37°C, pH 7.0, 30% dissolved oxygen, and an agitation speed of 80 rpm. The feed rate was adjusted daily to maintain a constant residual glucose concentration of 1.0–2.5 g L^–1. Product concentrations were determined using ELISA and are expressed as milligram product per liter of culture broth.

Figure 2

Fed batch culture supernatants after feed start and at the end of the process. Silver stained SDS–PAGE was used to analyze the total protein content in the culture supernatant. Western blot analysis was used to identify the recombinant proteins HSA (67 kDa) and 3D6scFv-Fc (homodimer: approx. 110 kDa), respectively. (A) Silver stained SDS–PAGE and (B) western blot under reducing conditions of the HSA producing CHO and P. pastoris clones. (C) Silver stained SDS–PAGE under non-reducing conditions, (D) Western blot under non-reducing, and (E) reducing conditions of the 3D6scFv-Fc expressing CHO and P. pastoris clones. Equal volumes of supernatant were loaded onto the gel. Samples of two independent cultivations were analyzed. The results of one representative replicate per clone are shown. Compared samples were always analyzed on the same gel.

Figure 3

Relationship between biomass-specific secretion rate q_P and specific growth rate μ during the feed phase of fed batch cultivations. (A) 3D6scFv-Fc and (B) HSA producing P. pastoris and CHO clones. P. pastoris and CHO cells were cultivated as described for Fig. 1. Product concentrations were determined using ELISA. Biomass-specific secretion rates were calculated using smoothed product concentrations (smoothing spline algorithm of the Matlab Curve Fitting Toolbox) of two independent cultivations. Specific growth rates represent mean values of two independent cultivations.